QIAsymphony SP Protocol Sheet

Transcription

1 QIAsymphony SP Protocol Sheet Purification of RNA from Tissues Using RNA_CT_400_V7, RNA_CT_800_V7, RNA_FT_400_V8, and mirna_ct_400_v8 General information These protocols are for purification of total RNA (>200 nt) or total RNA including mirna (and other small RNAs) from animal and human tissues using the QIAsymphony SP and the QIAsymphony RNA Kit. Depending on the sample type, we recommend using either the standard or large-volume protocol. Select RNA_CT_400_V7 (standard protocol) if processing up to 20 mg easy-to-lyse tissue or up to 10 mg thymus or spleen; select RNA_CT_800_V7 (large-volume protocol) if processing up to 50 mg easy-to-lyse tissue; select RNA_FT_400_V8 (fibrous tissue protocol) if processing up to 20 mg fibrous tissue; select mirna_ct_400_v8 to obtain total RNA including mirna from up to 20 mg of tissue (up to 10 mg thymus or spleen). Note: The RNA_FT_400_V8 and mirna_ct_400_v8 protocols should be used with QIAsymphony RNA Kits that have lot numbers higher than When using kits with lot number and lower, an inventory scan must be performed before first use with a new, unopened kit and the 200 µl elution volume cannot be used. If you have any questions, please contact QIAGEN Technical Services (see the back cover of our handbooks or visit Note: It is the user s responsibility to validate performance using this combination for any procedures used in their laboratory. February 2013 Sample & Assay Technologies

2 Standard protocol Kit QIAsymphony RNA Kit (cat. no ) Sample material Protocol name Default Assay Control Set Up to 20 mg easy-to-lyse tissue or up to 10 mg thymus or spleen RNA_CT_400_V7 ACS_RNA_CT_400_V7 Editable Elution volume: 50 µl, 100 µl, 200 µl Required software version Version 4.0 or higher Large-volume protocol Kit QIAsymphony RNA Kit (cat. no ) Sample material Protocol name Default Assay Control Set Up to 50 mg easy-to-lyse tissue or up to 30 mg thymus or spleen RNA_CT_800_V7 ACS_RNA_CT_800_V7 Editable Elution volume: 100 µl, 200 µl Required software version Version 4.0 or higher Fibrous tissue protocol Kit QIAsymphony RNA Kit (cat. no ) Sample material Protocol name Default Assay Control Set Up to 20 mg fibrous tissue RNA_FT_400_V8 ACS_RNA_FT_400_V8 Editable Elution volume: 50 µl, 100 µl, 200 µl Required software version Version 4.0 or higher QIAsymphony SP Protocol Sheet: RNA_CT_400, RNA_CT_800 (V7); RNA_FT_400, mirna_ct_400 (V8) page 2 of 9

3 mirna protocol Kit QIAsymphony RNA Kit (cat. no ) Sample material Protocol name Default Assay Control Set Up to 20 mg easy-to-lyse or fibrous tissue or up to 10 mg thymus or spleen mirna_ct_400_v8 ACS_miRNA_CT_400_V8 Editable Elution volume: 50 µl, 100 µl, 200 µl Required software version Version 4.0 or higher Sample drawer Sample type Sample amount Lysate volume Animal and human tissue Homogenate prepared from up to 50 mg of tissue, depending on protocol (see protocol tables above) 400 µl (standard, mirna, and fibrous tissue protocols) 800 µl (large-volume protocol) Primary sample tubes Secondary sample tubes n/a We recommend using 2 ml tubes (e.g., Sarstedt cat. no or ) or S-Blocks (cat. no ). For a full list of compatible vessels, see Inserts For more information, see the Resources tab at n/a = not applicable. QIAsymphony SP Protocol Sheet: RNA_CT_400, RNA_CT_800 (V7); RNA_FT_400, mirna_ct_400 (V8) page 3 of 9

4 Reagents and Consumables drawer Position A1 and/or A2 Position B1 Reagent cartridge n/a Tip rack holder 1 17 Disposable filter-tips, 200 µl or 1500 µl Unit box holder 1 4 Tip racks slots 5 and 12 Unit boxes containing sample prep cartridges or 8-Rod Covers Accessory troughs for ethanol n/a = not applicable. Waste drawer Unit box holder 1 4 Waste bag holder Liquid waste bottle holder Empty unit boxes Waste bag Empty liquid waste bottle Eluate drawer Elution rack (we recommend using slot 1, cooling position) For more information, see the Resources tab at QIAsymphony SP Protocol Sheet: RNA_CT_400, RNA_CT_800 (V7); RNA_FT_400, mirna_ct_400 (V8) page 4 of 9

5 Required plasticware RNA_CT_400_V7 RNA_CT_800_V7 RNA_FT_400_V8 mirna_ct_400_v Reagent cartridges Sample prep cartridges* 8-Rod Covers Disposable filter-tips, 1500 µl Disposable filter-tips, 200 µl Ethanol (ml) 70 2 x 2 x 70 2 x 2 x * 28 sample prep cartridges/unit box. Twelve 8-Rod Covers/unit box. 32 filter-tips/tip rack; the inventory scan requires additional tips (two 200 µl and seven 1500 µl tips). 96 samples per reagent cartridge; 72 samples in one run. Note: Numbers of filter-tips given may differ from the numbers displayed in the touchscreen depending on settings. We recommend loading the maximum possible number of tips. Elution volume The elution volume is selected in the touchscreen. Depending on the sample type and RNA content, the final eluate volume may vary by up to 15 µl less than the selected volume. Elution in smaller volumes increases the final RNA concentration, but reduces the yield and increases variability of the eluate volume. We recommend using the smallest elution volume only when the intended downstream application requires a higher RNA concentration. QIAsymphony SP Protocol Sheet: RNA_CT_400, RNA_CT_800 (V7); RNA_FT_400, mirna_ct_400 (V8) page 5 of 9

6 Preparation of sample material When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier. Important points before starting For best results, stabilize animal tissues immediately in RNAlater RNA Stabilization Reagent, or Allprotect Tissue Reagent. Tissues can be stored in the reagent at 37 C for up to 1 day, at C for up to 7 days, or at 2 8 C for up to 4 weeks (RNAlater) or 6 months (Allprotect). See the RNAlater Handbook or Allprotect Tissue Reagent Handbook for more information about the reagents and about stabilizing RNA in tissues. Buffer RLT Plus and buffers in the reagent cartridge contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach. See page 6 of the QIAsymphony RNA Handbook for safety information. Take appropriate safety measures and wear gloves when handling Buffer RLT Plus and reagent cartridges. QIAGEN Proteinase K (cat. no ) is required for the RNA_FT_400_V8 and mirna_ct_400_v8 protocols, but is not supplied with the QIAsymphony RNA Kit. It should be ordered separately. Things to do before starting Buffer RLT Plus may form a precipitate upon storage. If necessary, redissolve by warming (37 C) and then place at room temperature (15 25 C). -mercaptoethanol ( -ME) or dithiothreitol (DTT) must be added to Buffer RLT Plus before use to ensure RNA integrity. If using -ME, add 10 µl -ME per 1 ml Buffer RLT Plus. Dispense in a fume hood and wear appropriate protective clothing. -ME is stable in Buffer RLT Plus at room temperature for 1 month. Alternatively, add 20 µl of 2 M DTT per 1 ml Buffer RLT Plus. The stock solution of 2 M DTT in water should be prepared fresh or frozen in single-use aliquots. Buffer RLT Plus containing DTT can be stored at room temperature for up to 1 month. For isolation of total RNA including mirna using the mirna_ct_400_v8 protocol or the RNA_FT_400_V8 protocol, transfer 2 ml proteinase K solution to each of the tubes in positions 3 and 4 of the enzyme rack on the reagent cartridge (see Table 5 on page 23 of the QIAsymphony RNA Handbook). Note: Do not use <2 ml of proteinase K with a new cartridge. Tubes in positions 3 and 4 can remain empty with the lids on when using protocols that do not require proteinase K. Transfer 1.4 ml of DNase solution to each of the tubes in positions 1 and 2 of the enzyme rack on the reagent cartridge. For more information about preparation of DNase I, see the QIAsymphony RNA Handbook, page 25. QIAsymphony SP Protocol Sheet: RNA_CT_400, RNA_CT_800 (V7); RNA_FT_400, mirna_ct_400 (V8) page 6 of 9

7 For tissues with high DNA content, such as spleen and thymus, we recommend using 1 vial of DNase I to obtain 1.4 ml of DNase solution. An additional RNase-Free DNase Set (see ordering information, QIAsymphony RNA Handbook, page 42) will be required for each reagent cartridge used in that way. Tissues Fresh, frozen, or RNAlater or Allprotect stabilized tissue can be used for RNA purification. To freeze tissue for long-term storage, flash-freeze in liquid nitrogen and immediately transfer to 70 C (remove tissue from stabilization reagent before freezing). Tissue can be stored for several months at 70 C. To process, do not allow unstabilized tissue to thaw during weighing or handling before disruption in Buffer RLT Plus. Homogenized tissue lysates (in Buffer RLT Plus, step 3 of the procedure) can also be stored at 70 C for several months. To process frozen lysates, thaw samples at room temperature (15 25 C) or at 37 C in a water bath until they are completely thawed and salts in the lysis buffer are dissolved. Avoid extended treatment at 37 C, which can cause chemical degradation of the RNA. Continue with step 4 of the procedure. Pretreatment protocol for tissues 1. Excise the tissue sample from the animal or remove it from storage. Remove RNAlater or Allprotect stabilized tissues from the reagent using forceps. Remove any excess reagent. Do not use more than 20 mg tissue with the standard protocol or 50 mg with the high-volume protocol. Proceed immediately with step 2. Weighing tissue is the most accurate way to determine the amount. Note: For tissues of high cell density, such as thymus and spleen, do not use more than 10 mg for the standard protocol, or 30 mg for the high-volume protocol. 2. Follow either step 2a or 2b, depending on how the tissues were stabilized. 2a. For RNAlater or Allprotect stabilized tissues: If the entire piece of stabilized tissue can be used for RNA purification, place it directly into a suitably sized vessel for disruption and homogenization, and proceed with step 3. If only a portion of the stabilized tissue is to be used, place the tissue on a clean surface for cutting and cut it. Determine the weight of the piece to be used and place it into a suitably sized vessel for homogenization. Proceed with step 3. RNA in the RNAlater or Allprotect stabilized tissue is still protected while the tissue is processed at C. This allows cutting and weighing of tissues at ambient temperatures. It is not necessary to cut the tissue on ice or dry ice or in a refrigerated room. The remaining tissue can be placed into RNAlater or Allprotect reagent for further storage. Previously stabilized tissues can be stored at 70 C without the reagent. QIAsymphony SP Protocol Sheet: RNA_CT_400, RNA_CT_800 (V7); RNA_FT_400, mirna_ct_400 (V8) page 7 of 9

8 2b. For unstabilized fresh or frozen tissues: If the entire piece of tissue can be used for RNA purification, place it directly into a suitably sized vessel for disruption and homogenization, and proceed immediately with step 3. If only a portion of the tissue is to be used, determine the weight of the piece to be used, and place it into a suitably sized vessel for homogenization. Proceed immediately with step 3. RNA in tissues is not protected after harvesting until the sample is treated with RNAlater or Allprotect reagent, flash frozen, or disrupted and homogenized in protocol step 3. Frozen animal tissue should not be allowed to thaw during handling. The relevant procedures should be carried out as quickly as possible. Note: The remaining fresh tissue can be placed into RNAlater or Allprotect reagent for stabilization (see the RNAlater Handbook or the Allprotect Tissue Reagent Handbook). However, previously frozen tissue samples thaw too slowly in the reagent, thus preventing it from diffusing into the tissue quickly enough before the RNA begins to degrade. 3. Disrupt tissue and homogenize sample in Buffer RLT Plus. Disruption and homogenization of animal tissue can be performed by 2 alternative methods (3a or 3b). See Disruption and homogenization of starting material starting on page 20 of the QIAsymphony RNA Handbook for a more detailed description of disruption and homogenization methods. After storage in RNAlater RNA Stabilization Reagent or Allprotect Tissue Reagent, tissues may become slightly harder than fresh or thawed tissues. Disruption and homogenization of tissue samples using standard methods is usually not a problem. Note: Incomplete homogenization will lead to significantly reduced yields. Homogenization with rotor stator homogenizers or the TissueLyser II generally results in higher total RNA yields than with other homogenization methods. 3a. TissueLyser II homogenization: Place the weighed (fresh, frozen, or RNAlater-stabilized) tissue in a 2 ml microcentrifuge tube (not supplied), add 400 µl Buffer RLT Plus (800 µl for large-volume protocol), and add one stainless steel bead (3 7 mm diameter). Homogenize for 2 5 min at 25 Hz using the TissueLyser II. Rotate the TissueLyser rack, and homogenize for another 2 5 min at 25 Hz. Continue the protocol with step 4. Note: The instructions in step 3a are only guidelines. They may need to be changed depending on the sample being processed and on the bead mill being used. 3a. TissueRuptor homogenization: Place the weighed (fresh, frozen, or stabilized in RNAlater) tissue in a 2 ml microcentrifuge tube (not supplied). Add 400 µl Buffer RLT Plus (800 µl for largevolume protocol). Homogenize immediately using the TissueRuptor with a disposable probe until the sample is uniformly homogeneous (usually s).continue the protocol with step 4. Homogenization with the TissueRuptor simultaneously disrupts and homogenizes the sample. QIAsymphony SP Protocol Sheet: RNA_CT_400, RNA_CT_800 (V7); RNA_FT_400, mirna_ct_400 (V8) page 8 of 9

9 4. Centrifuge the tissue lysate for 3 min at maximum speed in a microcentrifuge. Carefully transfer the supernatant to sample tubes or plates that are compatible with the sample carrier of the QIAsymphony SP. For a full list of compatible labware, see We recommend using 2 ml tubes (e.g., Sarstedt, cat. no or ) or S-Blocks (see ordering information, in the QIAsymphony RNA Handbook, page 41). In some preparations, very small amounts of insoluble material will be present, making the pellet invisible. Lysates can stay on the instrument for 4 5 h at room temperature (15 25 C) without risk of RNA degradation. 5. For fatty tissues (e.g., brain, breast) only: add 100 µl chloroform to 400 µl lysate, and vortex thoroughly. Centrifuge at 4 C for 3 min at full speed ( 14,000 x g) to separate phases. Carefully transfer the aqueous phase to sample tubes or plates that are compatible with the sample rack of the QIAsymphony SP. 6. Place the tubes containing the lysates into the appropriate sample carrier, and load them into the Sample drawer. 7. Begin the purification process, as described in the General Purification Protocol. See the QIAsymphony RNA Handbook, page 24. For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at or can be requested from QIAGEN Technical Services or your local distributor. Trademarks: QIAGEN, Allprotect, QIAsymphony, TissueRuptor (QIAGEN Group); RNAlater (Ambion, Inc); Sarstedt (Sarstedt AG and Co.). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. RNAlater is a trademark of AMBION, Inc., Austin, Texas and is covered by various U.S. and foreign patents QIAGEN, all rights reserved. Australia Finland Japan Spain Austria France Korea (South) Sweden Belgium Germany Luxembourg Switzerland Brazil Hong Kong Mexico Taiwan Canada India The Netherlands UK China Ireland Norway USA Denmark Italy Singapore Sample & Assay Technologies